It is known that geminiviruses are a wide and diversified class of plant viruses that infect several plants of agronomic interest causing serious harvest losses. Such viruses are characterised by virions consisting of two geminate icosahedric particles. Their genome, consisting of one or two circular single-stranded DNA molecules (ssDNA), replicates in the nucleus of infected cells through double stranded intermediates (Hanley-Bowdoin et al., 1999).
The Geminiviridae family is divided in four genera named Mastrevirus, Begomovirus, Curtovirus and Topocuvirus based on the insect vector, the host spectrum and the genome structure (Briddon et al., 1985; Fauquet et al., 2003).
A serious disease of the tomato plant, transmitted by the whitefly Bemisia tabaci, is from a long time known as “tomato yellow leaf curl” in the areas of the Middle East, Asian South East and Africa, (Czosnek et al., 1997). This disease, that can cause harvest losses of 100% (Picò et al., 1996; Czosnek et al., 1997), successively spread both throughout the Western Mediterranean, reaching Sardinia, Sicily and Spain (Czosnek et al., 1997), and America (Polston et al., 1997).
Recently the agents of the disease have been identified and isolated, being viruses belonging to the Geminiviridae family, genera Begomovirus. Phylogenetic studies have highlighted the presence of different viral species related to different geographical origins of the Begomovirus: Asia, Africa and America (Czosnek et al., 1997).
The genoma of the Tomato yellow leaf curl Sardinia virus (TYLCSV) species, is monopartite (Kheyr-Pour et al., 1991). The DNA is transcribed bidirectionally and contains six open reading frame (ORF), two on the viral strand (V): V1 and V2, and four on the complementary strand (C): C1, C2, C3 and C4, as shown in FIG. 1. Between the C1 and V2 ORFs there is a non-coding region named intergenic region (IR) analogous to that present in the genome of all Geminiviridae. The genomic organization of TYLCSV is structurally similar to that of the bipartite Begomoviruses component A such as the tomato golden mosaic virus (TGMV) and the African cassava virus (ACMV). In the case of bipartite Begomoviruses the nomenclature of the ORFs present on the component A of the complementary strand is: AL1 or AC1, AL2 or AC2, AL3 or AC3, AL4 or AC4, while on the viral strand AR1 or AV1, AR2 or AV2; on the complementary strand of the component B is: BL1 or BC1 and on the viral strand BR1 or BV1.
Strategies used until now in order to control the infection of the geminiviruses transmitted by the Bemisia tabaci are based on the use of expensive fine mesh nets (for the cultivation of fresh-market tomato) and particularly on repeated insecticide treatments (cultivations of both fresh-market and processing tomato). Such strategies result in an increase of the production expenses and represent a serious danger for the health of the agricultural operators and consumer. Furthermore the onset of Bemisia tabaci populations resistant to the insecticide imidacloprid has been already reported (Cahill et al., 1996; Williams et al., 1996).
The development of resistant cultivated species represents the most practical and economic way to control viral infections. Classical breeding programs for introducing resistance to geminiviruses that cause the tomato yellow leaf curl were based on the transfer of resistance genes from wild species of Lycopersicon to species of cultivated tomato. Thereby lines with variable levels of resistance to TYLCSV have been obtained and commercialized, the best lines showing reduced symptoms and low viral replication. However plants with low and mean levels of resistance represent a potential receptacle for further infections.
Another important aspect to be considered is that the agronomic characteristics of the lines obtained are not always optimal and however reflects those of the genotype of cultivated tomato used in breeding programs.
A tomato line immune to the viruses causing the tomato yellow leaf curl disease, namely, with neither symptoms nor viral DNA replication has not been released yet.
With the advent of genetic engineering new perspectives were opened up for the introduction of resistance characters against plant viruses. Most strategies are based on the introduction and expression of pathogen-derived sequences in the plant of interest, Pathogen Derived Resistance (PDR) (Sanford & Johnson, 1985; Abel et al., 1986; Tavazza and Lucioli, 1993).
Although such strategies have been successfully applied for the introduction of resistance characters to plant viruses with RNA genome (Beachy, 1997), in the case of geminiviruses, with a DNA genome, the expression of pathogen-derived sequences has produced plants with no lasting resistance and/or tolerance.
The mechanisms that induce virus resistance achieved through the expression of pathogen-derived sequences can be grouped in two wide classes:                a) resistance mediated by the expression of a pathogen protein such as, for instance, the expression of a dominant negative mutant;        b) resistance mediated by the post-transcriptional gene silencing (Baulcombe, 1996; Beachy, 1997; Zaitlin and Palukaitis, 2000).        
The post-trascriptional gene silencing is a ubiquitary process in eukaryotes, involving the degradation of specific RNAs following the formation of double strand RNA (dsRNA) molecules having sequences homologous to the target RNA.
Although there may be different contexts able to induce the production of dsRNA homologous to the transgene (transcription of aberrant transgenic RNAs, presence in the transgenic RNA of sufficiently long inverted and repeated sequences, integration of the transgene in the plant genome in inverted and repeated multiple copies), once the dsRNA is produced, the latter is recognised and degraded in short molecules of dsRNA of about 21-26 nucleotides, referred to as siRNA.
The siRNAs are then integrated in a multiprotein complex named RISC, that is able to degrade all RNAs having sequence homology with the siRNAs. The latter ones represent therefore the determining factors of RNA silencing specificity and their presence related to a determined sequence establishes univocally that this RNA sequence is post-transcriptionally silenced.
Therefore, transgenic plants post-transcriptionally silenced for sequences derived from viral RNA genome, are resistant to the homologous virus and to viruses with nucleotide sequences closely related to the transgene.
The transgene silencing can be also induced following virus infection.
In fact, viral replication is able to induce silencing of a transgene, initially not silenced, if the nucleotide sequence of the transgene is homologous to a portion of the infecting virus genome. The activation of the silencing mechanism involves the specific degradation of the RNA molecules having sequence homology with the inducer RNA.
As direct consequence, the silencing activation by the virus is associated with a degradation of both transgenic mRNA sequences homologous to the virus and viral genome. This results in the host recovery after an initial infectious step, so that the new vegetative part is proved to be virus free. A peculiar characteristic of the plant tissues that develop subsequently to the recovery phenomenon is that they are highly resistant to a following infection by the same virus.
The resistance mediated by post-transcriptional gene silencing, since based on recognition at the nucleotidic level, confers resistance only against viral isolates closely homologous to the virus genome from which the transgene was derived. Instead, strategies based on the expression of a pathogen protein normally produce plants resistant also to viral strains or isolates not-closely related from a nucleotide point of view.
It is also been shown that the transgene silencing is influenced by the temperature, being inactive at temperatures below 15° C. (Szittya et al., 2003). Therefore plants exposed in field conditions at temperature range below 15° C. can lose the silencing-mediated resistance.
It must be borne in mind that, although from several years transgenic plants resistant to RNA genome viruses have been achieved through mechanisms based on transgene silencing, so far it is not reported that such strategy can be successfully applied to the geminiviruses (DNA genome-viruses).
It's clear that the best strategy in order to obtain plants resistant to a wide spectrum of geminiviruses is the one in which the interfering product is the protein. It is clear that the width of the resistance spectrum increases the agronomic and commercial value of the produced plant.
Thereby the expression in transgenic plants of dysfunctional variants of geminivirus replicative Rep protein has been used in order to obtain plants with greater levels of resistance or immunity against the geminiviruses.
It's known in literature that the expression of a truncated replicative Rep protein (Rep-210) of TYLCSV is able to confer resistance against viral infection, although such resistance is not lasting because the virus is able to overcome it over time.
In tables 1 and 2 are shown the results of the analysis of the resistance of TYLCSV-agroinoculated Rep-210 expressing transgenic plants of Tomato 47×wt (Brunetti et al. 1997) and of N. benthamiana line 102.22 (Noris et al. 1996) respectively.
TABLE 1No infected% infectedLycopersiconTimeplants/inoculatedplants/inoculatedesculentum(weeks)plantsplantsRep-21040/13092/1315185/1338Wild-type46/6 100
TABLE 2No infected% infectedNicotianaTimeplants/inoculatedplants/inoculatedbenthamiana(weeks)plantsplantsRep-2102 4/2119311/2152418/2186Wild-type26/6100
From the results reported in tables 1 and 2, it can be clearly inferred that the resistance against TYLCSV mediated by the transgenic expression of a pathogen-derived sequence, is overcome with time.
Similarly, also the resistance induced by the transgenic expression of a dominant negative mutant of Rep of the bipartite geminivirus “African Cassava Mosaic Virus” is overcome with time (Sangaré et al., 1999).
Another example is represented by the transgenic expression of the TYLCV capsid protein in a tomato interspecific hybrid (Lycopersicon esculentum X L. pennellii) which confers a partial resistance against viral infection (Kunik et al., 1994). Even in this case the resistance mediated by the expression of the capsid protein is not long lasting and it results to be poorly useful from an agronomic point of view.
In the light of the above, is clear the need to have new methods that would allow to use successfully the polynucleotide sequences derived from the geminiviruses in order to obtain long lasting resistant plants against geminiviruses.
The authors of the present invention have now prepared polynucleotide sequences encoding pathogen-derived viral proteins and able to confer virus resistance to the host, suitably modified in order to be ineffective targets of the virus-induced post-transcriptional gene silencing to obtain transgenic plants with lasting levels of resistance against geminiviruses.
In fact during the experiments the authors show that the overcoming of the resistance, and therefore the difficulty to achieve lasting resistance against geminiviruses, is due to the unexpected abilities of the geminiviruses to silence post-transcriptionally the transgene and to spread in a plant in which the transgene, with sequences homologous to the infecting virus, is post-transcriptionally silenced.
As shown in FIGS. 2 and 3, respectively, both in the transgenic plants of N. benthamiana line 102.22 and in the plants of Tomato 47×wt the virus ability to overcome the resistance results from the transgene silencing by the same virus and from the unexpected ability of the virus to spread in a silenced plant.
The TYLCSV ability to spread in a plant in which the transgene Rep-210 is post-transcriptionally silenced, is further circumstantiated as set forth in FIG. 4.
The results show that the transgenic tomato plants 47×10D (Brunetti et al., 1997), post-transcriptionally silenced before agroinoculation, as shown by the absence of the Rep-210 protein and by the concurrent presence of the transgene-homologous siRNAs, are susceptible to the TYLCSV infection as well as the controls.
From the above it results that, contrary to RNA viruses, the geminivirus is not blocked by an active silencing of viral gene sequences. The above said is not limited to the kind of transgenic plant to be used or the way the virus should be inoculated, through agroinfection or Bemisia tabaci. In fact, as shown in table 3, using a reduced number of viruliferous bemisia per plant, so as to infect between 90% and 100% of the control plants, about 40% of transgenic plants (line 201) whose transgene is post-trascriptionally silenced, are not or late infected, while at a higher inoculum concentration, all the plants challenged with viruliferous insects are infected similarly to the experiments carried out using agroinoculation.
TABLE 3Low concentrationHigh concentrationMolecular Analysisof inoculumaof inoculumbbefore inoculum2c36236Transgenic plantsRep-210siRNAs 6/15 7/15 8/1516/2120/2121/21(No)(Si)Not transgenicRep-210siRNAs11/1211/1211/128/88/88/8(No)(No)aSeven viruliferous insects per plant for 2 daysbThirty-five viruliferous insects per plant for 5 dayscWeeks after inoculum
Therefore it's important to consider that the viral agroinoculation conditions used for testing the resistance and assessing persistence over time (as shown in FIGS. 2, 3 and 4 and in tables 1 and 2) correspond to high or very high viral pressure conditions. This experimental approach allows to identify transgenic plants with very high resistance levels or immune against the viral infection and therefore of very high commercial value.
Accordingly, the introduction of resistance characters against geminiviruses through the expression of pathogen-derived sequences is limited due to the unexpected ability of the geminiviruses to silence post-trascriptionally the transgene and to spread in the silenced plant.
Furthermore the authors show that the transcripts both of positive (V1 and V2) and negative strand (C1, C2, C3 and C4) of TYLCSV are subjected, during a normal infection on wild-type plants, to the viral post-trascriptional silencing, as shown in FIG. 5. This results in the impossibility to achieve long-term resistance through expression of sequences derived from the same pathogen, unless these are suitably modified in order not to be a target or to be an ineffective target of the virus-induced post-trascriptional gene silencing. Instead, by introducing in the plant genome a sequence suitably mutated or chosen according to the invention it is possible to obtain a long lasting resistance against geminiviruses, unlike that achieved with the known methods.